The goal of this project is to elucidate the mechanism by which active and inactive transcription is established, organized, and maintained within the inactive X-chromosome (Xi) in mammalian females. Orchestration of gene expression and repression is crucial for proper development and cell viability. X-chromosome inactivation (X-inactivation), a mechanism to dosage compensate X-linked genes between XY males and XX females in mammals, is an example of programmed gene regulation whereby one of the two X-chromosomes is transcriptionally inactivated in early female embryos. Several non-coding genes localize to the X-inactivation center (Xic), and their interplay initiates X-inactivation. Whle one of the two X-chromosomes in females is inactivated in a chromosome-wide fashion, expression from escape genes on the Xi is essential for proper female development in humans because haploinsufficiency of escape genes affects embryonic survival and causes Turner syndrome in live-born females. Thus, a better understanding of the molecular mechanism underlying gene regulation on the Xi is of high clinical relevance. However, little is known about how organized gene expression and repression are established within the Xi. In this proposal, we aim to dissect the molecular basis of how transcriptionally active compartments are established in an otherwise transcriptionally inactive environment. Our central hypothesis is that escape genes on the Xi are regulated in two different ways: protection from X-inactivation and transcriptional activation. In addition to the conventional boundary elements and/or genomic context model, both published studies and our preliminary data indicate that escape gene reactivation is an essential step for escape gene expression from the Xi. Based on our preliminary studies using a mouse embryonic stem (ES) cell system, we propose the Xi-specific reactivation model for escape gene expression on the Xi. We will investigate the mechanism of escape gene expression by (Aim 1) determining how chromatin modifiers that promote transcriptional activation regulate escape gene expression on the Xi, (Aim 2) elucidating the role of the 3 end of Xist RNA in X-linked gene regulation during X-inactivation, and (Aim 3) using our novel functional assay system to dissect how genetic elements contribute to create transcriptionally active compartments. The proposed study in gene regulation during X-inactivation will potentially reveal a universal role for long non-protein coding (non-coding) RNAs in a wide variety of biological processes. PUBLIC HEALTH RELEVANCE: The proposed study will uncover the molecular mechanism of X-linked gene regulation via long non-coding RNAs during X-chromosome inactivation, which is essential for embryonic viability and proper development for mammalian females. Thus, the proposed study has relevance for both birth defects and genetic disorders, such as cancer in humans, and is thus of high clinical significance. Because long non-coding RNAs are involved in a wide variety of critical biological processes (including imprinting and tumor suppression), an understanding of non-coding RNA-mediated gene regulation will lead to an understanding of the genetic causes of imprinted gene disorders, such as Beckwith-Wiedemann and Angelman syndromes and cancer.